Department of Genetics, Faculty of Medicine, University of Porto, Porto, Portugal.
Epigenetics. 2011 Nov;6(11):1354-61. doi: 10.4161/epi.6.11.17993. Epub 2011 Nov 1.
Paternal imprinting marks were shown to be erased in the mouse primordial germ cells and progressively re-established throughout the male germ line development, starting in fetal prospermatogonia and continuing post-natally through the onset of meiosis. We here evaluated imprinting marks in human adult spermatogenic cells and analyzed mRNA and protein expression of DNA Methyltransferases (DNMTs). Spermatogonia A, primary and secondary spermatocytes, round spermatids and elongated spermatids/spermatozoa were isolated by micromanipulation from testicular biopsies of men with normal spermatogenesis. DNA methylation at two imprinted genes, H19 and MEST/PEG1, was analyzed using bisulphite genomic sequencing and DNMTs expression was determined by qRT-PCR and immunofluorescence. H19 was completely methylated at the spermatogonia stage in the analyzed individuals and MEST/PEG1 was completely demethylated, with the exception of few CpGs. The analysis of DNMT1, DNMT3A and 3B expression showed peaks of mRNA transcripts in primary spermatocytes and in mature ejaculated spermatozoa, with DNMT1 transcript level being the most abundant in all cell stages. Immunolocalization showed that DNMT proteins are present throughout the spermatogenic cycle, with stage-specific shuttling between the nucleus and cytoplasm. We conclude that, in humans, methylation imprints are established in spermatogonia A and are maintained in subsequent stages up to elongated spermatid/spermatozoa. Additionally, DNA methyltransferases are expressed throughout human spermatogenesis, possibly maintaining the methylation patterns in order to avoid the transmission of imprinting errors by the male gamete.
父系印迹标记被证明在小鼠原始生殖细胞中被抹去，并在雄性生殖系发育过程中逐渐重新建立，从胎儿生殖细胞开始，并在出生后通过减数分裂的开始继续。我们在这里评估了人类成年精原细胞中的印迹标记，并分析了 DNA 甲基转移酶（DNMTs）的 mRNA 和蛋白表达。通过显微操作从具有正常精子发生的男性睾丸活检中分离出精原细胞 A、初级和次级精母细胞、圆形精子和伸长精子/精子。使用亚硫酸氢盐基因组测序分析了两个印迹基因 H19 和 MEST/PEG1 的 DNA 甲基化，通过 qRT-PCR 和免疫荧光测定 DNMTs 的表达。在所分析的个体中，H19 在精原细胞阶段完全甲基化，MEST/PEG1 完全去甲基化，除了少数 CpG 外。DNMT1、DNMT3A 和 3B 表达的分析显示，mRNA 转录本在初级精母细胞和成熟射出的精子中出现峰值，DNMT1 转录本水平在所有细胞阶段最为丰富。免疫定位显示，DNMT 蛋白存在于整个精子发生周期中，在细胞核和细胞质之间有特定的穿梭。我们得出结论，在人类中，甲基化印迹在精原细胞 A 中建立，并在随后的阶段中保持，直到伸长精子/精子。此外，DNA 甲基转移酶在人类精子发生过程中表达，可能维持甲基化模式，以避免雄性配子传递印迹错误。
